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. 2014 Mar 24;592(Pt 12):2549–2561. doi: 10.1113/jphysiol.2013.268680

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice

Rachel A Gioscia-Ryan 1, Thomas J LaRocca 1, Amy L Sindler 1, Melanie C Zigler 1, Michael P Murphy 2, Douglas R Seals 1
PMCID: PMC4080937  PMID: 24665093

Abstract

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD.

Key points

  • The development of age-related arterial endothelial dysfunction, a key antecedent of increased cardiovascular disease (CVD) risk, is mediated largely by reduced nitric oxide bioavailability as a consequence of oxidative stress.

  • Mitochondria are critical signalling organelles in the vasculature, which, when dysregulated, become a source of excessive reactive oxygen species; the role of mitochondria-derived oxidative stress in age-related vascular dysfunction is unknown.

  • We show that a mitochondria-targeted antioxidant, MitoQ, ameliorates vascular endothelial dysfunction in old mice and that these improvements are associated with the normalization of mitochondria-derived oxidative stress and markers of arterial mitochondrial health.

  • These results indicate that mitochondria-derived oxidative stress is an important mechanism underlying the development of age-related vascular endothelial dysfunction and therefore may be a promising therapeutic target.

  • Mitochondria-targeted antioxidants represent a novel strategy for preserving healthy vascular endothelial function in primary ageing and preventing age-related CVD in humans.

Introduction

Advancing age is the primary risk factor for cardiovascular disease (CVD) (Roger et al. 2012), which is driven significantly by the development of arterial endothelial dysfunction (Yeboah et al. 2007; Herrera et al. 2010). Impaired endothelium-dependent dilation (EDD), largely caused by reduced nitric oxide (NO) bioavailability secondary to increased oxidative stress, is an important clinical manifestation of endothelial dysfunction (van der Loo et al. 2000; Lakatta, 2003; Bachschmid et al. 2013).

Oxidative stress, a state in which the production of pro-oxidant molecules such as superoxide outweighs endogenous antioxidant defence mechanisms, directly and indirectly reduces NO bioavailability. Superoxide reacts with NO to form peroxynitrite, which, in turn, can oxidize and inactivate tetrahydrobiopterin, a necessary co-factor for endothelial NO synthase (eNOS). As a result, eNOS becomes uncoupled and produces additional superoxide rather than NO (van der Loo et al. 2000; Brandes et al. 2005). Peroxynitrite also propagates oxidative stress via excessive nitration of cellular proteins, including the key antioxidant enzyme manganese superoxide dismutase (MnSOD), effectively reducing endogenous antioxidant capacity (van der Loo et al. 2000; Brandes et al. 2005). Overall, the age-related increase in oxidative stress represents a vicious cycle of processes that interact to reduce NO and impair endothelial function.

Healthy mitochondria are crucial for maintaining numerous aspects of physiological function; mitochondrial reactive oxygen species (mtROS), primarily produced as superoxide and subsequently converted to hydrogen peroxide, play a crucial role in cellular signalling and the maintenance of homeostasis in the vasculature (Quintero et al. 2006; Dai et al. 2012; Dromparis & Michelakis, 2013; Kluge et al. 2013). However, excessive mtROS production leads to a state of cellular oxidative stress and is a hallmark of mitochondrial dysfunction (Murphy, 2009; Dromparis & Michelakis, 2013). Excess mtROS may promote the disruption of vascular function directly (via oxidative stress) and indirectly through mitochondrial dysfunction-induced alterations in signalling (Dai et al. 2012; Dromparis & Michelakis, 2013; Kluge et al. 2013). Mice with genetic MnSOD deficiency, a model of elevated mitochondrial oxidative stress, display impairments in endothelial function (Wenzel et al. 2008). However, the role of mtROS in primary ageing-associated vascular endothelial dysfunction has not been established.

Compounds that specifically target mtROS hold great promise for ameliorating impairments, including vascular dysfunction, that stem from mitochondria-associated oxidative stress (Cochemé et al. 2007; Murphy & Smith, 2007; Lakshminarasimhan & Steegborn, 2011; Smith et al. 2012). Because mitochondrial oxidative stress is closely linked to overall mitochondrial health, reducing mitochondrial oxidative stress may have the potential to restore homeostasis in this critical cellular organelle and, in turn, improve cellular homeostasis and physiological function. In contrast to traditional non-targeted exogenous antioxidants, which have been ineffective in clinical trials (Kris-Etherton et al. 2004), mitochondria-targeted antioxidants accumulate specifically at this site of ROS production and may, therefore, be more effective in reducing oxidative stress and the resulting functional sequelae (Cochemé et al. 2007; Smith et al. 2012). One such compound is mitochondria-targeted ubiquinone, or MitoQ. MitoQ is a biochemically modified form of the naturally occurring antioxidant ubiquinone conjugated to a lipophilic cation [decyl-triphenylphosphonium (TPP)] (Murphy & Smith, 2007; Smith & Murphy, 2010). The positive charge and lipophilicity of this cation drive MitoQ to accumulate predominately on the inner mitochondrial membrane (Ross et al. 2008), where it blocks mitochondrial oxidative damage. The reduced form of MitoQ is then regenerated via reaction with mitochondrial respiratory complex II (succinate-coenzyme Q reductase) (Ross et al. 2008; Smith & Murphy, 2010). MitoQ has been studied in models of CVD and in patients with clinical disease, but the efficacy of MitoQ in reversing primary arterial ageing, including vascular endothelial dysfunction, is unknown.

Here, we tested the hypothesis that oral MitoQ treatment would ameliorate vascular endothelial dysfunction in old mice, and that these improvements would be associated with reductions in vascular mitochondrial oxidative stress and improvements in vascular mitochondrial health.

Methods

Ethical approval

All studies were approved by the Institutional Animal Care and Use Committee at the University of Colorado Boulder and conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, USA; 2011).

Animals

Male c57BL/6 mice, which represent an established model of age-related vascular endothelial dysfunction (Sprott & Ramirez, 1997; Brown et al. 2007), were purchased from the ageing colony at the National Institute on Aging (National Institutes of Health, Bethesda, MD, USA) at ∼6 months or ∼25 months of age and allowed to acclimate to our facilities for 2 weeks prior to beginning treatment. Mice were housed in standard cages on a 12 : 12 h light : dark cycle and were allowed access to normal rodent chow (Harlan 7917) and water ad libitum. Body mass and water intake were monitored regularly throughout the study.

MitoQ treatment

Based on previous findings of effective doses and durations of treatment of MitoQ (Rodriguez-Cuenca et al. 2010; Smith & Murphy, 2010), mice were randomly assigned to treatment with MitoQ (250 μm) [young MitoQ-treated mice (YMQ), ∼8 months, n = 6; old MitoQ-treated mice (OMQ), ∼27 months, n = 14] or normal drinking water [young control mice (YC), ∼8 months, n = 12; old control mice (OC), ∼27 months, n = 13] for 4 weeks. MitoQ (Antipodean Pharmaceuticals, Inc., Menlo Park, CA, USA; gifted by M.P.M.) was prepared fresh and administered in light-protected water bottles that were changed every 3 days. To rule out potential effects of the TPP cation (mitochondria-targeting moiety), additional groups of young (YMP) and old (OMP) mice were provided with drinking water containing a control compound comprising only decyl-TPP cation (n = 5 or 6 per group) and not the antioxidant (Adlam et al. 2005; Graham et al. 2009; Smith & Murphy, 2010).

Vascular endothelial function

Following the 4 week treatment period, mice were anaesthetized with isofluorane and killed by exsanguination via cardiac puncture. EDD and endothelium-independent dilation (EID) were measured in isolated carotid arteries as previously described (Rippe et al. 2010; LaRocca et al. 2013). The carotid arteries were dissected free of surrounding tissue and cannulated onto glass micropipettes in warmed (37°C) physiological saline solution in myograph chambers (Danish Myo Technology A/S, Aarhus, Denmark). Arteries were pressurized to 50 mmHg and allowed to equilibrate for ∼1 h prior to the beginning of experiments. Following preconstriction with phenylephrine (2 μm; Sigma-Aldrich Corp., St Louis, MO, USA), EDD was assessed by measuring the increase in luminal diameter in response to increasing concentrations of acetylcholine (Ach; 1 × 10−9 m to 1 × 10−4 m; Sigma-Aldrich Corp.). EID was measured as dilation in response to increasing doses of the exogenous NO donor sodium nitroprusside (SNP; 1 × 10−10 m to 1 × 10−4 m; Sigma-Aldrich Corp.). To account for baseline differences in vessel diameter, all dose–response data are reported on a percentage basis.

NO-mediated EDD

EDD was assessed in the presence of the eNOS inhibitor N-nitro-l-arginine methyl ester (l-NAME; 0.1 mm, 30 min incubation; Sigma-Aldrich Corp.) and the contribution of NO was calculated as the difference between maximal dilation to ACh alone and maximal dilation in the presence of l-NAME (Rippe et al. 2010; LaRocca et al. 2012).

Tonic mtROS suppression of EDD

To determine tonic mtROS suppression of EDD, arteries were incubated for 40 min with 1 μm MitoQ to scavenge mtROS prior to assessment of EDD to ACh as described above.

Acute mtROS challenge

To determine the effects of an acute increase in mtROS on EDD, a subset of arteries was incubated for 40 min with 0.5 μm rotenone (Sigma-Aldrich Corp.), a concentration previously shown to stimulate mtROS production at respiratory Complex I without completely inhibiting cellular respiration (Weir et al. 1991; Li et al. 2003), prior to assessment of EDD to ACh. The difference between maximal dilation to ACh in the presence versus absence of rotenone was calculated to determine the rotenone-induced decrement in EDD.

Aortic whole-cell and mitochondria-specific superoxide production

Measurement of superoxide production in the thoracic aorta was performed using electron paramagnetic resonance spectroscopy, as described previously (Fleenor et al. 2012; LaRocca et al. 2012, 2013). Briefly, the aorta was removed and dissected free of surrounding tissue. Segments of 2 mm were incubated for 1 h at 37°C in Krebs-Hepes buffer with the superoxide-specific spin probe 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (0.5 mm; Enzo Life Sciences, Inc., Farmington, NY, USA) or mitochondrial superoxide-specific spin probe MitoTEMPO-H (0.5 mm; Enzo Life Sciences, Inc.) for detection of whole-cell and mitochondria-specific superoxide production, respectively (Dikalova et al. 2010; Dikalov et al. 2011). The signal amplitude was analysed using an MS300 X-band EPR spectrometer (Magnettech GmbH, Berlin, Germany) with the following settings: centrefield, 3350 G; sweep, 80 G; microwave modulation, 3000 mG, and microwave attenuation, 7 dB. Data are presented relative to the mean of the young control group.

Aortic protein expression

Protein expression was determined using standard Western blotting techniques in segments of thoracic aorta, a representative large elastic artery that, unlike the carotid, provides sufficient sample for analysis (Rippe et al. 2010; LaRocca et al. 2012). Following homogenization in radio-immunoprecipitation assay lysis buffer, 15 μg of aortic protein were loaded onto 4–12% polyacrylamide gels and transferred onto nitrocellulose membranes (Criterion System; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were incubated (overnight at 4°C) with primary antibodies: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1 : 1000, normalizer; Cell Signaling Technology, Inc., Boston, MA, USA); phosphorylated (ser36) p66SHC (1 : 500; Abcam, Inc., Cambridge, MA, USA); cytochrome c oxidase subunit IV (COX IV; 1 : 1000; Cell Signaling Technology, Inc.); MnSOD (1 : 2000; Enzo Life Sciences, Inc.); nitrotyrosine (NT; 1 : 500; Abcam, Inc.), and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; 1 : 1000; Cell Signaling Technology, Inc.). Proteins were visualized on a digital acquisition system (ChemiDoc-It; UVP, Inc., Upland, CA, USA) using chemilluminescence with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Westgrove, PA, USA) and enhanced chemilluminescence (ECL) substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). Relative intensity was quantified using ImageJ software (National Institutes of Health) and normalized to GAPDH intensity and then expressed as a ratio of the mean intensity of the young control group.

Nitric oxide synthase activity assay

Activity of nitric oxide synthase (NOS) was determined in aortic lysates using the Ultrasensitive Colorimetric Assay for Nitric Oxide Synthase (Oxford Biomedical Research, Rochester Hills, MI, USA), according to the manufacturer's directions. Briefly, 30 μl of aortic lysate (containing 40–60 μg protein) were incubated with NOS substrates NADPH and l-arginine for 4 h to allow continual production of NO by NOS. This reaction was carried out in aqueous solution, in which NO rapidly degrades into the more stable products nitrate and nitrite. Nitrate reductase was added to the samples to facilitate the enzymatic conversion of nitrate to nitrite. Subsequently, nitrite (representing the total NO generated by NOS) was quantified colorimetrically using Griess reagent. The concentration of nitrite in each sample was determined by interpolating from a standard curve generated using known concentrations of nitrite. Values were normalized to the protein content of each sample and expressed as μmoles of NO produced per μg of aortic protein.

Statistical analysis

All statistical analyses were performed using IBM spss Statistics for Windows Version 21.0 (IBM Corp., Armonk, NY, USA) with an α-value of 0.05. EDD and EID dose responses to ACh and SNP, respectively, were analysed using two-factor (treatment group × dose) repeated-measures ANOVA. Within-group differences in EDD dose responses to ACh in the absence versus presence of pharmacological modulation (e.g. l-NAME, rotenone) were also determined using two-factor (condition × dose) repeated-measures ANOVA. For all other outcomes, group differences were determined using one-way ANOVA. When a significant main effect was observed, Tukey's honestly significant difference post hoc tests were performed to determine specific pairwise differences.

Results

Animal characteristics and MitoQ intake

Selected morphological characteristics and water intake are shown in Table 1. There were no differences in body mass across groups, and organ weights did not differ between control and MitoQ-treated mice, indicating an absence of off-target effects. MitoQ intake in young and old treated groups was similar.

Table 1.

General morphological characteristics and MitoQ intake

YC OC YMQ OMQ
Body mass (g) 30.06 ± 2.15 30.38 ± 3.69 28.97 ± 0.80 29.51 ± 1.54
Heart mass (mg) 151 ± 11 184 ± 37* 142 ± 22 178 ± 10*
Liver mass (g) 1.75 ± 0.16 1.62 ± 0.31 1.40 ± 0.12 1.22 ± 0.28
Quadriceps mass (mg) 171 ± 3.2 146 ± 18* 199 ± 31 135 ± 25*
Visceral fat mass (mg) 478 ± 208 335 ± 229 384 ± 162 288 ± 266
MitoQ intake (μmol day−1) N/A N/A 1.07 ± 0.33 1.01 ± 0.28
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Data are presented as group means ± s.e.m. Abbreviations: YC, young (∼8 months) control mice; OC, old (∼27 months) control mice; YMQ, young (∼8 months) MitoQ-supplemented mice; OMQ, old (∼27 months) MitoQ-supplemented mice; N/A, not applicable.

*

P < 0.05 vs. YC and YMQ.

MitoQ treatment reverses the age-related decline in EDD

Primary comparison

Carotid artery dose response (Fig. 1A) and peak (Fig. 1C) EDD to ACh were reduced in old compared with young mice. In vivo (4 weeks) MitoQ supplementation restored EDD in old mice (Fig. 1A and C).

Figure 1. MitoQ reverses the age-related decline in endothelium-dependent dilation.

Figure 1

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Endothelium-dependent dilation (EDD) dose responses to acetylcholine (ACh) in carotid arteries. A, primary group comparisons of young (YC) and old (OC) control mice provided with normal drinking water and old mice supplemented with MitoQ (OMQ). B, control group comparisons of young (YMP) and old (OMP) mice treated with decyl-triphenylphosphonium (TPP) and young mice supplemented with MitoQ (YMQ) (*P < 0.05 vs. YC; main effect of group). C, maximal EDD to ACh (*P < 0.05 vs. YC). D, endothelium-independent dilation to the nitric oxide donor sodium nitroprusside. Data are presented on a percentage basis to account for differences in vessel diameter among groups. Values are means ± s.e.m. (n = 6–13/group).

Control comparisons

MitoQ treatment had no effect on EDD in young mice (Fig. 1B and C) and EDD did not differ between young or older control (normal drinking water) and decyl-TPP-treated mice (Fig. 1B and C).

There were no differences in EID in response to the NO donor SNP among the groups (Fig. 1D). These results indicate that short-term treatment with MitoQ restores ACh-mediated endothelial function in ageing mice. Because the normal drinking water and decyl-TPP treatment groups did not differ, all subsequent analyses were performed only in the normal drinking water and MitoQ treatment groups (YC, OC, YMQ and OMQ) in order to increase statistical power.

MitoQ treatment restores NO bioavailability in old mice

The impairment in EDD in old mice was a result of reduced NO bioavailability, as indicated by a lesser reduction in EDD upon incubation with the eNOS inhibitor l-NAME (Fig. 2A and B). MitoQ supplementation normalized the NO component of EDD in old mice and had no effect in young mice (Fig. 2B). These results indicate that MitoQ increases NO bioavailability and restores the NO component of EDD in old mice.

Figure 2. MitoQ restores nitric oxide-dependent endothelium-dependent dilation in old mice.

Figure 2

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A, endothelium-dependent dilation (EDD) dose responses to acetylcholine (ACh) in the absence/presence of the nitric oxide (NO) inhibitor N-nitro-l-arginine methyl ester (l-NAME) in carotid arteries of young (YC) and old (OC) control mice and young (YMQ) and old (OMQ) MitoQ-supplemented mice. Data are presented on a percentage basis to account for differences in vessel diameter among groups. (*P < 0.05 vs. YC, main effect of group for dose response to ACh alone; #P < 0.05 within-group, dose response to Ach + l-NAME vs. dose response to ACh alone.) There were no group differences in the dose response to ACh + l-NAME. B, NO-dependent dilation (maxEDDACh–maxEDDACh+l-NAME) (*P < 0.05 vs. YC). C, total NOS activity in the aorta (*P < 0.05 vs. YC). All data are presented as means ± s.e.m. (n = 6–8/group).

Total NOS activity was reduced in arteries of old compared with young mice; this was not altered by MitoQ supplementation (Fig. 2C), indicating that the restoration of NO bioavailability with MitoQ treatment did not result from changes in eNOS activity.

MitoQ supplementation normalizes vascular oxidative stress in old mice

Aorta from old mice exhibited greatly increased NT (Fig. 3A), a biomarker of general oxidative protein modification (Radi, 2004, 2013), and markedly increased whole-vessel superoxide production (Fig. 3B) compared with those from young mice. MitoQ treatment ameliorated the age-related increases in aortic NT and superoxide and had no effect in young mice. These data indicate that MitoQ has potent antioxidant effects in arteries of old mice, which may contribute to its beneficial effects on vascular endothelial function.

Figure 3. MitoQ normalizes vascular oxidative stress in old mice.

Figure 3

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A, nitrotyrosine (NT), a biomarker of oxidative protein damage, in arteries (aorta) of young (YC) and old (OC) control mice and young (YMQ) and old (OMQ) MitoQ-supplemented mice; representative Western blot images (25 kDa and 55 kDa bands) are shown below. B, whole-cell superoxide production in aortic segments; representative electron paramagnetic resonance spectra are shown below. Protein expression data are normalized to GAPDH expression. All data are normalized to YC mean values and presented as means ± s.e.m. (n = 5–8/group; *P < 0.05 vs. YC).

MitoQ supplementation reverses arterial mitochondria-derived oxidative stress and suppression of function in old mice

Aortic mitochondrial superoxide production was substantially greater in old compared with young mice (Fig. 4A), as was aortic phosphorylated (activated) p66SHC (Fig. 4B), a signalling protein that is a marker and master regulator of mitochondrial oxidative stress (Gertz & Steegborn, 2010). MitoQ treatment in old mice ameliorated the age-related increases in mitochondrial superoxide and p-p66SHC.

Figure 4. MitoQ reduces arterial mitochondria-derived oxidative stress and suppression of function in old mice.

Figure 4

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A, mitochondria-specific superoxide production in aortic segments of young (YC) and old (OC) control mice and young (YMQ) and old (OMQ) MitoQ-supplemented mice; representative electron paramagnetic resonance spectra are shown below. B, aortic protein expression of phosphorylated (serine36) p66SHC; representative Western blot images are shown below. C, dose response and maximal (inset) endothelium-dependent dilation to acetylcholine in the presence of MitoQ (1.0 μm, 40 min incubation to scavenge mitochondrial reactive oxygen species). Protein expression data are normalized to GAPDH expression. Protein expression and mitochondrial superoxide data are normalized to YC mean values. All values are presented as means ± s.e.m. (n = 6–8/group; *P < 0.05 vs. YC).

Acute (40 min) incubation of carotid arteries with MitoQ (1.0 μm) restored EDD to ACh in old control animals, indicating excessive mitochondrial superoxide-mediated suppression of EDD with ageing (Fig. 4C). Acute MitoQ treatment had no effect on EDD in young control or young and old MitoQ-treated animals.

Together, these results indicate that an increase in arterial mitochondrial superoxide production and mitochondrial oxidative stress contributes to age-related declines in endothelial function. Moreover, the data suggest that MitoQ treatment markedly reduces vascular mitochondrial superoxide production and oxidative stress in a process associated with the complete rescue of endothelial function in old mice.

MitoQ restores markers of vascular mitochondrial health in old mice

Protein expression of markers of mitochondrial signalling/biogenesis, antioxidant defence and mass (PGC-1α, MnSOD and COX IV) was reduced in arteries of old compared with young mice (Fig. 5A–C). MitoQ treatment restored these markers of mitochondrial health in old mice to levels similar to those in young controls, and further increased COX IV expression in young mice. These results indicate that reducing mitochondrial oxidative stress with MitoQ treatment may also restore mitochondrial homeostasis in the ageing vasculature.

Figure 5. MitoQ improves markers of mitochondrial health in old mice.

Figure 5

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Protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) (A), manganese superoxide dismutase (MnSOD) (B) and cytochrome c oxidase subunit IV (COX IV) (C) in aorta of young (YC) and old (OC) control mice and young (YMQ) and old (OMQ) MitoQ-supplemented mice; representative Western blot images are shown below. Protein expression data are normalized to GAPDH expression and YC mean values and presented as means ± s.e.m. (n = 6–10/group; *P < 0.05 vs. YC).

MitoQ supplementation improves resistance to acute mtROS stress in arteries of old mice

Acute incubation with 0.5 μm rotenone to stimulate production of mtROS (Weir et al. 1991; Li et al. 2003) caused a significant further (∼25%) reduction in carotid artery EDD to ACh in old control mice, but had no significant effect on EDD in young control mice (Fig. 6). MitoQ treatment attenuated the rotenone-induced impairment of EDD in old mice, but had no effect in young mice. These results indicate that ageing is associated with reduced resistance to an acute mtROS challenge in arteries of mice, and that MitoQ treatment improves resistance to this mtROS stressor to levels observed in young mice.

Figure 6. MitoQ improves resistance to acute mtROS stress in arteries of old mice.

Figure 6

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Endothelium-dependent dilation (EDD) dose responses to acetylcholine (ACh) in the absence/presence of rotenone (0.5 μl, 40 min incubation to induce mitochondrial superoxide production) in carotid arteries of young control (YC) and MitoQ-supplemented (YMQ) mice (A) and old control (OC) and MitoQ-supplemented (OMQ) mice (B) (n = 5–6/group). Data are presented on a percentage basis to account for differences in vessel diameter among groups. Data for young and old mice are presented separately for clarity. [*P < 0.05 within-group, dose response to Ach + rotenone vs. dose response to ACh alone; #P < 0.05 vs. OMQ (main effect of group) for dose response to ACh alone.] C, impairment in EDD induced by acute incubation with rotenone (maxEDDACh–maxEDDACh+ROTENONE). Data are presented as means ± s.e.m. (n = 5–6/group; *P < 0.05 vs. within-group maximal dilation to ACh alone).

Discussion

The present results demonstrate for the first time that a mitochondria-targeted antioxidant (MitoQ) completely reverses endothelial dysfunction in old mice, restores NO bioavailability and normalizes total and mitochondria-derived arterial superoxide production and oxidative stress (NT abundance). These improvements are accompanied by the normalization of age-related declines in markers of vascular mitochondrial health, and an increased ability of arteries to resist acute stress induced by excessive mtROS.

The present results extend previous findings by our laboratory showing that restoring NO bioavailability and reducing oxidative stress in old mice ameliorates endothelial dysfunction (Rippe et al. 2010; Sindler et al. 2011; Donato et al. 2013; Fleenor et al. 2013; LaRocca et al. 2013) by specifically examining the role of mitochondria-derived oxidative stress and dysregulation in mediating age-related endothelial dysfunction. Although it is well established that age-related declines in mitochondrial function and increases in mitochondria-derived oxidative stress contribute to the development of dysfunction in other tissues such as skeletal muscle, heart and brain (Sastre, 2003; Balaban et al. 2005; Weber & Reichert, 2010), the role of vascular mitochondrial oxidative stress in mediating impairments in endothelial function in primary ageing is unknown. Our results demonstrate that ageing is associated with increases in mitochondria-specific superoxide production and protein markers of mitochondrial oxidative stress in the large elastic arteries of mice. Importantly, inhibiting mitochondrial oxidative stress with MitoQ either acutely (ex vivo) or chronically (in vivo supplementation) abolished the age-related reduction in EDD by restoring NO bioavailability secondary to a reduction in oxidative stress, and not by obvious improvement in eNOS enzyme activation or function. These observations provide strong evidence that excess mitochondrial oxidative stress is an important mechanism underlying the development of endothelial dysfunction with ageing, and support the apparent efficacy of mitochondria-targeted strategies to improve endothelial function in ageing.

Mitochondrial production of ROS has previously been implicated in the progression of vascular dysfunction in the settings of clinical CVD and in genetic models of mitochondrial antioxidant deficiency. Production of mtROS can be induced by exposing cultured endothelial cells to adverse conditions associated with cardiometabolic disease (e.g. hyperglycaemia) (Shenouda et al. 2011), and cross-sectional studies in humans and rodent models have shown that CVD is accompanied by increased vascular mitochondrial damage/dysfunction (Ballinger, 2002; Zhang & Gutterman, 2007; Ungvari et al. 2008). Endothelial function is also impaired in mice with genetic MnSOD insufficiency, a model of excess mitochondrial oxidative stress (Wenzel et al. 2008). Together, data in experimental and disease models indicate that excess mtROS play a critical role in mediating vascular dysfunction (Wenzel et al. 2008). However, the present data provide the first evidence that mtROS contribute to the vascular endothelial dysfunction associated with primary ageing.

Despite the relative paucity of mitochondria in the endothelium compared with tissues such as skeletal muscle and liver (Blouin et al. 1977), our results suggest a pivotal role of mitochondria-related signalling and mtROS in modulating endothelial function with age. This possibility is further supported by previous studies showing a life-extending effect of endothelial cell-specific knockout of p66SHC, a signalling protein involved in sensing and regulation of mtROS production (Camici et al. 2007; Gertz & Steegborn, 2010). We observed a marked elevation in phosphorylation of p66SHC, an indication of its activation (Gertz & Steegborn, 2010), in the arteries of old compared with young mice, and this was accompanied by increased vascular mitochondrial superoxide production. MitoQ normalized p66SHC activation and reduced mitochondrial superoxide production, suggesting that an increase in mtROS-mediated vascular oxidative stress may be a key mechanism contributing to the age-related decline in endothelial function.

Our results also support a critical role for healthy mitochondria in the maintenance of vascular endothelial function in ageing. Mitochondria are an important source of cellular ROS, which are produced at several sites, including Complexes I, II and III of the respiratory chain, enzymes involved in electron transfer and mitochondrial metabolism (e.g. α-ketoglutarate dehydrogenase, electron transfer flavoprotein : coenzyme-Q oxidoreductase), p66SHC, the monoamine oxidase family of enzymes on the outer mitochondrial membrane, and nicotinamide adenine dinucleotide phosphate oxidase 4, which localizes to the mitochondria (Murphy, 2009; Kluge et al. 2013). Because of their role in the production of and their proximity to ROS, mitochondria are particularly vulnerable to oxidative damage (Murphy, 2009; Weber & Reichert, 2010). Healthy mitochondria are equipped with antioxidant defence systems that act to maintain mtROS at physiological levels and facilitate signalling functions (Nunnari & Suomalainen, 2012; Dromparis & Michelakis, 2013), but prolonged, uncontrolled oxidative stress can lead to the inhibition of mitochondrial function and related signalling pathways, including a reduction in mitochondrial antioxidant enzymes and biogenesis (Anderson & Prolla, 2009; Galluzzi et al. 2012; Dromparis & Michelakis, 2013). Our findings indicate that the age-related increase in vascular mtROS is associated with the disruption of vascular mitochondrial homeostasis, as we observed a decline in the mitochondrial antioxidant MnSOD in the arteries of old mice, as well as reductions in protein markers of mitochondrial biogenesis (PGC-1α) and mass (COX IV), all of which were restored with MitoQ treatment.

Mitochondria are crucial for mediating the cellular response to oxidative stress, such as occurs cumulatively with advancing age (Sastre, 2003; Zhang & Gutterman, 2007; Galluzzi et al. 2012; Nunnari & Suomalainen, 2012). Our observation of the impaired ability of arteries in old mice to resist acute mtROS-induced stress (administration of rotenone) is indicative of age-related dysregulation of mitochondria, including a reduction in mitochondrial antioxidant defences (e.g. MnSOD). This finding is consistent with previous work showing that genetic MnSOD deficiency in mice aggravates age-associated endothelial dysfunction (Wenzel et al. 2008). Rotenone, a known inhibitor of mitochondrial respiratory Complex I, also stimulates mtROS production without completely inhibiting cellular respiration when applied at low concentrations (<1 μm) (Li et al. 2003). Previous studies (Csiszar et al. 2006; Griffith et al. 1986; Rodman et al. 1991; Weir et al. 1991) examining the effects of mitochondrial respiratory inhibition on endothelial function have demonstrated impairment in EDD following rotenone administration (at concentrations of ≥1 μm), but the magnitude of impairment differed among species and types of arteries tested. It is also plausible that complete inhibition of mitochondrial respiration may have distinctly different effects on vascular function than an increase in mtROS, which may perhaps explain the lack of significant impairment observed in our young mice, which, in contrast to old mice, are expected to have mitochondrial antioxidant defences adequate to counter this acute stressor. Importantly, MitoQ treatment in old mice restored the ability of arteries to resist an acute increase in mtROS to levels similar to that observed in young mice. Together, our data provide evidence that reducing mitochondrial oxidative stress in the vasculature (e.g. via MitoQ supplementation) may restore overall mitochondrial health, with corresponding functional improvements.

In support of the present observations in primary ageing, MitoQ has also been reported to ameliorate dysfunction associated with mitochondrial oxidative damage in several animal models of clinical disease, including cardiac ischaemia–reperfusion injury, sepsis, fatty liver disease, kidney disease, neurodegeneration and CVD (reviewed in Smith & Murphy, 2010). Previously, MitoQ supplementation initiated prior to the establishment of CVD in young (8 weeks of age) stroke-prone hypertensive rats was found to prevent the development of endothelial dysfunction (Graham et al. 2009); however, the present results are the first to demonstrate the efficacy of MitoQ in reversing vascular dysfunction in aged animals. MitoQ treatment has also been well tolerated in Phase II clinical trials in patients with liver and neurological diseases (Gane et al. 2010; Snow et al. 2010), which underscores its strong potential for translation to treatments of human vascular ageing.

Although the observed changes in arterial mitochondrial superoxide production and protein expression of select mitochondrial markers strongly suggest age- and treatment-associated effects on mitochondrial health and homeostasis, future investigation is warranted to more fully characterize vascular mitochondrial function (e.g. respiratory function, ATP production, calcium signalling, biogenesis, fission/fusion dynamics) in this setting. It is well established that mtROS and mitochondrial dysfunction contribute to adverse cellular signalling, including activation of inflammatory pathways that may exacerbate endothelial dysfunction (Ungvari et al. 2007; Nunnari & Suomalainen, 2012; López-Armada et al. 2013), but the potential for mitochondrial antioxidant treatment to attenuate adverse inflammatory signalling in the vasculature is currently unknown. Our findings that MitoQ treatment not only reduces mitochondrial oxidative stress but also restores markers of mitochondrial health in arteries of old mice indicate that this treatment may reduce age-related adverse signalling via the restoration of mitochondrial homeostasis.

In conclusion, the present study demonstrates for the first time that a mitochondria-targeted antioxidant, MitoQ, effectively reverses age-related vascular endothelial dysfunction, restores NO bioavailability, normalizes total and mitochondria-derived oxidative stress, and improves vascular mitochondrial health and stress resistance. These results indicate that mitochondria-targeted antioxidants may represent a novel promising strategy for the preservation of healthy vascular endothelial function in primary ageing and the prevention of age-related CVD in humans (Fig. 7).

Figure 7. Working hypothesis.

Figure 7

Open in a new tab

An increase in vascular mitochondria-derived reactive oxygen species (mtROS) production and associated dysregulation of mitochondrial homeostasis with primary ageing contributes to a state of oxidative stress and a reduction in nitric oxide (NO) bioavailability, which promote the development of endothelial dysfunction. Mitochondria-targeted antioxidant treatment with MitoQ may be a promising therapeutic strategy for reducing vascular mitochondrial oxidative stress, restoring vascular mitochondrial homeostasis, and preserving endothelial function in advancing age to reduce cardiovascular disease (CVD) risk.

Acknowledgments

The authors would like to thank Micah Battson for his technical assistance.

Glossary

ACh

acetylcholine

CVD

cardiovascular disease

COX IV

cytochrome c oxidase subunit IV

EDD

endothelium-dependent dilation

EID

endothelium-independent dilation

eNOS

endothelial nitric oxide synthase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

MnSOD

manganese superoxide dismutase

mtROS

mitochondrial reactive oxygen species

l-NAME

Nω-nitro-l-arginine methyl ester

NO

nitric oxide

NOS

nitric oxide synthase

NT

nitrotyrosine

PGC-1α

peroxisome proliferator-activated receptor-γ coactivator-1α

SNP

sodium nitroprusside

TPP

triphenylphosphonium

Additional information

Competing interests

M.P.M. is on the scientific advisory board of Antipodean Pharmaceuticals, Inc. All other authors declare that they have no competing interests.

Author contributions

R.A.G.-R., T.J.L., M.P.M. and D.R.S. contributed to the conception and design of the experiments. R.A.G.-R., T.J.L., A.L.S., M.C.Z. and D.R.S. contributed to the collection, analysis and interpretation of data. R.A.G. and D.R.S. contributed to the drafting and critical revision of the article. All authors provided final approval of the submitted version. All experiments were performed at the University of Colorado Boulder.

Funding

This work was supported by the National Institutes of Health (AG000279, AG039210, AG013038).

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